Establishment Of An ATCase-based Protein Fusion Expression System

As the implementation of life,proteins carrying out the most basic life functions.in the 21 st age of technology,biotechnology is rapidly developed accompanying the evolution of various theories and methods,but there is still some tricky problem in the field of protein annotation.These problems include,but are not limited to,low expression level,the formation of inclusion body,low stability,and least likely to crystallize.Thus,researchers have proposed corresponding solutions to these problems,such as,add a small molecule ubiquitin to improve the solubility and stability,add a green fluorescent protein(GFP)label for orientation.Aspartate Carbamoyltransferase,short for ATCase,is a multi-subunit ‘petal-like' protein complex composed of 12 subunits with 2 trimers of catalytic subunits and 3 dimers of regulatory subunits.In this study,we have constructed a new expression vector combined p ET-15b-sumo with the regulatory subunits of ATCase,named as p ET-15b-SUMO-ATC.This new vector would theoretically contribute to the dimerization of the target protein.To test the practicability of this new vector,telomerase TERT protein was expressed and purified.Using dynamic light scattering(DLS),size exclusion chromatography(SEC),and small-angle X-ray scattering(SAXS)to examine the aggregation state of the fused TERT protein,we obtained the following results.1,We have got a fused TERT protein in high purity and proved that the fused protein was dimerized using DLS and SEC.Moreover,the primer extension and SAXS experiment show that the fused TERT protein was folded properly and maintained its activities.2,Since ATCase is a 12 subunits complex,did this characteristic possessed generality for a fused ATC-TERT? We have mixed the ATC-TERT and the catalytic subunits(ATC-SC)in vitro to observe weather a hetero-dodecamer could emerge.DLS and SEC results demonstrated that ATC-TERT and ATC-SC formed a heterododecamer successfully.3,More proteins,like De Pif1 and DEAD-box protein,were constructed into p ET-15b-SUMO-ATC to further test the universality of this new fusion vector.Experiment results prove that all these proteins can be dimerized,and all the fused protein could form a hetero-dodecamer after incubation with ATC-SC protein in vitro.4,All the experiment proves that we have obtained a new expression vector that could help dimerized the target protein yet without altering their original folding states and maintain their activities.This method could be helpful for obtaining a protein with an appropriate size that is suitable for electron microscope observation.Besides,the formation of polymers is also useful to improve the stability of some small proteins.